Many medical and research applications exist today for compounds labelled with stable isotopes. For example, in the areas of structure determination and the elucidation of metabolic pathways, deuterium, .sup.13 C or .sup.15 N-labelled macromolecules can play essential roles. Labelling with stable isotopes has been used in determining the structure of recombinant proteins (see, for example, Torchia, D. A., et al., J. Am. Chem. Soc. 110:2320 [1988]) and the structure and metabolism of polysaccharides from such microorganisms as Streptococcus species and Pseudomonas species (see Wessels, M. R., et al., J. Biol. Chem. 262:8262 [1987] and Knirel, Y. A. et al., Eur. J. Biochem. 166:189 [1987]). Labelling with stable isotopes also has been used in determining the structure and biosynthesis of antibiotics (see, for example, Beutler, J. A., et al., J. Natl. Proc. Acad. Sci. 51:562 [1988]) and in the biosynthesis of labeled tracers, such as amino acids (see Walker, T. E. and R. E. London, Appl. Env. Microbiol. 53:92 [1987]).
A practical and convenient way in which to label compounds and macromolecules produced by various microorganisms is to culture the microorganism in a growth medium which contains one or more nutrients labelled with the stable isotope of interest. For example, perdeuterated glucose, which is available commercially, is an excellent substrate for preparing perdeuterated E. coli. The high cost of perdeuterated glucose, however, is such that its use is not viable for inexpensive commercial, large-scale media for culturing cells. Accordingly, other labelled substrates capable of supporting growth of desired microorganisms, plant cells and mammalian cells have been sought.
One focus of the investigation has been on algal-based growth media. The terms "algae" and "algal-based" are used herein to denote microalgae, unless otherwise noted. Algal-based growth media have been found to be excellent sources of nutrients for bacteria, able to support rapid growth. Algae grow photosynthetically, with CO.sub.2 as the sole carbon source and H.sub.2 O as the sole source of hydrogen for cellular material. Several species of algae, such as Chlorella vulgaris, Chlorella pyrenoidosa and Scenedesmus obliquus, have been shown to grow in 99.9% D.sub.2 O (defined herein as pure D.sub.2 O) with inorganic salts and CO.sub.2 as the source of carbon. Under these conditions, deuterium replaces all of the hydrogen in the algae. See, for example, Taecker, R. G., et al., Biotechnol. Bioeng. 13:779 (1971). Algae also can grow using 99.9% .sup.13 CO.sub.2 as their carbon source (Behrens, et al., Journal Applied Phycology, [1989]) or using Na.sup.15 NO.sub.3 or K.sup.15 NO.sub.3 in place of NaNO.sub.3 or KNO.sub.3, respectively, as their nitrogen source. As algae are rich in proteins and carbohydrates, algae grown under such conditions incorporate .sup.2 H, .sup.13 C or .sup.13 N uniformly as the corresponding component in the cells. See, for example, Crespi, H. L. et al., Nature 184:729 (1959).
The first report in the literature of the development of algal-based growth media for culturing and labelling heterotrophic organisms with stable isotopes was by Blake et al., J. Pharm. Sci. 50:425 (1961). Their general procedure for the production of a deuterated-algal-based growth medium involves slurrying deuterated algae with deuterium oxide and then adding the slurry to boiling deuterium oxide to rupture the algal cell walls and release the cellular components. The solution then is cooled and centrifuged, and the residue is extracted with a mixture of methanol and petroleum ether to remove pigments and lipids. The insoluble fraction is dried, then hydrolyzed by refluxing with 1N deuterium chloride for 24 hours. The solution is filtered, the residue discarded, and the hydrolysate then is treated with silver carbonate to remove chloride. The solution is centrifuged and the supernatant is passed through an ion exchange column. The column is washed to remove the nonionic fraction, primarily glucose and mannose. The ionic fraction, comprised primarily of amino acids, then is removed from the column by eluting with 1N HCl.
Although the hydrolysate obtained using this procedure has proved to be a useful medium for culturing and labelling various organisms, further improvements have been sought. The procedure has not been used to make a commercial product, thus researchers wishing to use algal-based growth media must prepare their own growth media from purchased labelled raw materials. Also, the procedure developed by Blake et al. is disadvantageous in that it is expensive, time-consuming and gives inconsistent results according to different laboratory practices. Furthermore, efforts to culture cells of various microorganisms on media prepared in accordance with this procedure often have been unsatisfactory; cell growth has been very limited. Accordingly, it would be highly advantageous to have ready to use, characterized and labelled algal-based growth media that can be inoculated directly by the researcher.
It is an object of this invention to provide a labelled algal-based growth medium that can be used directly by researchers to culture and label various cells and the compounds and macromolecules they produce.
It is a further object of this invention to produce an algal-based growth medium that can support efficient cellular growth.
Further objects of this invention will become apparent from reading the following description of the invention.